7030
Biochemistry 1993, 32, 7030-7034
Calcium ATPase of Sarcoplasmic Reticulum Has Four Binding Sites for Calcium? William P. Jencks,' Tong Yang, Daniel Peisach, and Jayhyuk Myung Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110 Received March 30, 1993
ABSTRACT: The calcium-transporting ATPase of sarcoplasmic reticulum is known to bind two Ca2+ ions from the cytoplasm to the free enzyme and two Ca2+ions from the lumen to the phosphoenzyme. The concentration of phosphoenzyme formed at equilibrium from Pi and Mg2+ increases with increasing concentration of calcium in the lumen, which binds to the phosphoenzyme to form Ca2.E-PeMg. However, at subsaturating concentrations of Mg2+ increasing the concentration of lumenal Ca2+ does not drive phosphoenzyme formation to completion. The maximal levels of phosphoenzyme that are formed at saturating concentrations of lumenal Ca2+increase with increasing concentrations of Mg2+. This result requires that Ca2+ can bind to low-affinity lumenal sites on both the free enzyme and the phosphoenzyme, as well as to the high-affinity cytoplasmic calcium-binding sites. If there were no lumenal binding sites for Ca2+on the free enzyme, high concentrations of lumenal Ca2+would convert all of the enzyme to the same maximal concentration of Ca2-E-P-Mg at subsaturating concentrations of Mg2+ and Pi. We conclude that there are two low-affinity lumenal sites as well as two high-affinity cytoplasmic sites for Ca2+on the free enzyme. Phosphorylation by ATP results in translocation of Ca2+ from the high-affinity to the low-affinity sites.
The coupling between the hydrolysis of ATP and the internalization of two Ca2+ions that is brought about by the Ca2+-ATPase1 of sarcoplasmic reticulum vesicles can be defined by a simple set of rules that describe changes in chemical and vectorial specificity,as shown in Scheme 1.The chemical specificity for catalysis of phosphorylation by ATP or Pi is controlled by the presence or absence of bound Ca2+ ions, while the vectorial specificity for Ca2+ binding or dissociation on the two sides of the membrane is controlled by the state of phosphorylation of the enzyme (Pickart & Jencks, 1984). This scheme was first described by Makinose (1973). The mechanism by which the two Ca2+ ions are transported across the membrane is of particular interest. It is well-known that two Ca2+ ions bind to high-affinity sites of the unphosphorylated enzyme on the cytoplasmic side of the sarcoplasmic reticulum membrane and to low-affinity sites on the lumenal side of the phosphoenzyme (Makinose & Hasselbach, 1965; Carvalho & Leo, 1967; Dupont, 1978; Kalbitzer et al., 1978);Le., phosphorylation acts as a vectorial switch that changes the side of the membrane at which calcium binds and dissociates during the transport process. Phosphorylation also decreases the affinity of the enzyme for calcium: the dissociationconstants for the two Ca2+ions from the unphosphorylated enzyme are in the micromolar range, while they are in the millimolar range for the phosphoenzyme (Makinose & Hasselbach, 1965; Carvalho & Leo, 1967; Dupont, 1978;Kalbitzer et al., 1978;Pickart & Jencks, 1984). There are at least two different ways in which these changes in calcium binding and dissociation could occur: (1) There can be a phosphorylation-dependent change in gating of the binding sites for two Ca2+ions, such that calcium at these sites can bind and dissociate on the cytoplasmic side 7 ContributionNo.1753. Thisresearchwassupportedinpartby grants from the National Institutes of Health (GM20888) and the National Science Foundation (DMB-8715832). 1 Abbreviations: Ca**-ATPase,calcium-transportingATPase; EGTA, ethylene glycol bis(&aminoethyl ether)-N,N,N',N'-tetraaccticacid; EP, phosphoenzyme; MOPS, 3-(N-morpholino)propanesulfonic acid; Pi, inorganic phosphate; SR, sarcoplasmic reticulum; SRV, sarcoplasmic reticulum vesicles; Tris, tris(hydroxymethy1)aminomethane.
Scheme I
of the membrane with the unphosphorylated enzyme, E, and on thelumenal side of the membrane with the phosphoenzyme (Figure 1A). (2) Calcium on the unphosphorylatedenzyme can dissociate from one pair of binding sites to the cytoplasmic side of the membrane, while calcium bound to the phosphorylatedenzyme can dissociate to the lumenal side of the membrane from a different set of binding sites (Figure 1B). According to this mechanism, phosphorylation brings about a translocation of the two calcium ions from one set of binding sites to another, whereas accordingto the first mechanismthere is only a change in the gating of a single pair of binding sites upon phosphorylation. Several investigators (Makinose & Hasselbach, 1965; Dupont, 1978; Chaloub et al., 1979) and notably Suko et al. (198 1) have proposed that low-affinity calcium-binding sites exist on the lumenal side of the membrane in the free enzyme, as well as the phosphoenzyme. This conclusion is based upon an extensive series of measurements of the concentration of phosphoenyzme that is formed at equilibrium in the presence of different concentrations of Mg2+and Pi with vesicles that were passively loaded with 30 or 40 mM calcium (Suko et al., 1981). It was shown that characteristic patterns for the dependence of phosphoenzyme concentration on the concentration of Mg2+ and Pi are expected if there are lumenal calcium binding sites in the free enzyme and these binding sites are saturated with calcium. The results of these
0006-2960/93/0432-7030$04.00/0 0 1993 American Chemical Society
Biochemistry, Vol. 32, No. 27, I993 7031
Four Ca2+-Binding Sites on the CaZ+-ATPaseof SR Cytoplasm &
1 .o
,P
I
/ A
I
0.0
Lumen
,:
,
0
10
20
30
40
0
10
20
30
40
Cytoplasm
/
1 Lumen
B FIGURE1: Schematic models for the binding and dissociation of two Ca2+ ions on the cytoplasmic side of the membrane with the free enzyme and the lumenal side of the membrane with the phosphoenzyme. (A) A two-site model, with a change in gating upon phosphorylation. (B) A four-site model, in which the two Ca2+ ions are translocated from high- to low-affinitysites upon phosphorylation,
experiments, especially the finding that the dependence of the concentration of phosphoenzyme on the concentration of MgZ+ at equilibrium does not differ significantly at 30 and 40 mM lumenal Ca2+, are consistent with the existence of lumenal Caz+-binding sites on the free enzyme that are saturated with calcium. However, it is difficult to becertain that a sufficiently large range of calcium concentrations was examined in these experiments to establish the existence of interior binding sites beyond question. We undertook a reinvestigation of this problem because of the importance of the question of whether lumenal binding sites for Ca2+ exist on the free enzyme, in addition to the cytoplasmic sites, and because we wished to determine what concentration of lumenal calcium is required to saturate these sites, if they do exist. We report here what we believe to be simple and direct evidence in support of the existence of low-affinity lumenal binding sites for calcium on the free enzyme. If there are no such lumenal binding sites, the binding of calcium to form Ca2-E-P-Mg can be described by
[Gal (mW
FIGURE2: (A) If there are no lumenal binding sites for Ca2+ in the free enzyme, increasingthe concentration of lumenal Ca*+at different concentrations of Mg2+ or Pi will drive phosphoenzyme formation to completion. (B) If there are lumenal binding sites for Ca2+ in the free enzyme, increasing the concentration of lumenal Ca2+ will drive phosphoenzyme formation to completion when the concentrations of Me3+and Pi are saturating but will give lower maximal concentrations of phosphoenzyme in the presence of subsaturating concentrations of Mg2+ and Pi. The curves were calculated from eqs 3 and 4 for the mechanisms of eqs 1 and 2, respectively, with KOI= 5 mM, K02 = 6 mM, K l = K2 = 10 mM, K3 = K4 = 1 mM, K, = 1.0, Ks = K7 = 1 mM, [Pi] = 2 mM, and [Mg2+] = 0.05 0.25 (- - -), and 10 (-) mM. (-e),
FD.
We have derived eqs 3 and 4 for the reaction schemes shown in eqs 1 and 2, respectively. If two Ca2+ ions bind to the
Etot -ZEP-
(
+
+ 1) K K KOlKOZ +[Gal KO1
+ K4[Pi]+ K,[Mg] + [Mg][Pi] (4)
According to this mechanism, phosphoenzyme formation can be driven to completion by high concentrations of lumenal calcium at subsaturating concentrations of MgZ+ and Pi (Figure 2A). If there are lumenal Ca*+-binding sites on the free enzyme, the reaction is described by eq 2. With this mechanism, an increase in the concentration of lumenal Ca2+will not drive the reaction to completion at subsaturating concentrations of Mg2+and Pi, because Ca2+ can bind to the lumenal sites of both the free enzyme and the phosphoenzyme (Figure 2B).
lumenal sites of the free enzyme, with dissociation constants Kol and KOZ(eq 2), the concentration of lumenal calcium, [Ca], appears in both the numerator and denominator of eq 4 and an increase in the lumenal calcium concentration will not force all of the enzyme into the species Ca2.E-P-Mg. However, if there is no binding of lumenal calcium to the free enzyme and, therefore, no Kol and KOZterms (eq l), the term for calcium binding inside the vesicle appears only in the denominator of eq 3 and a high concentration of lumenal
7032 Biochemistry, Vol. 32, No. 27, 1993
Jencks et al.
Table I: Dependence of Phosphoenzyme Concentration at Equilibrium on the Concentrations of Magnesium and Lumenal Calcium' phosphoenzyme (nmol/mg) [Ca2+]lumen[Mg2+]0.25 [Mgz+]0.5 [Mg2+]1.0 [Mg2+]10 mM mM mM (mM) mM 0 0 2 2 5 5 10 10 15 15 23 23 31 31 40 40
0.1 1 0.1 1 0.33 0.33 0.60 0.61 0.81 0.82 0.72 0.83 0.99 0.99 1.22 1.29 1.13 1.11
0.20 0.20 0.56 0.58 0.90 0.90 1.19 1.17 1.20 1.25 1.38 1.40 1.68 1.62 1.58 1.57
0.31 0.29 0.75 0.76 1.24 1.26 1.67 1.69 1.65 1.58 1.75 1.75 1.98 2.05 1.74 1.72
0.67 0.64 1.47 1.52 1.80 1.88 2.34 2.33 2.00 2.05 2.19 2.24 2.51 2.45 2.29 2.34
a Vesicles passively loaded with calcium were added to a solution containing 2 mM 32P-labeledinorganic phosphate and the indicated concentrations of MgC12, 30 mM KCI, 10 mM EGTA, and 40 mM MOPS/Tris buffer, pH 7.0, at 25 OC. The amount of phosphoenzyme formationwas determined after 25 s by precipitationwith acid, filtration, and scintillation counting, as described in the text.
calcium will convert all of the enzyme to CayE-P.Mg. Equation 4 reduces to eq 3 when the concentration of lumenal Ca2+ is
